Regulation Of HCV Replication By Cellular Proteins Interacting With 3' Non-coding Region Of HCV Genome

The 3' non-coding regeion(NCR) of the hepatitis C virus(HCV) is believed to function in the initiation and regulation of viral RNA replication and protein translation by interacting with the viral and host components.To examine host proteins interacting with the HCV 3'NCR,biotinylated 3'NCR,and its reverse complementary 5'(-)NCR were used in RNA pull-down assay.Cellular proteins from Huh7 cells pulled down by biotinylated RNAs were identified by 2DE/MALDI-TOF MS and 1DE/LC/MS methods.Totally,10 proteins could be identified from both methods,among which six bound specifically to the 3'NCR,three proteins to the 5'(-)NCR only,and one protein bound to both.Three identified proteins(PCBP2, G3BP1,and DDX1) were selected for further investigation into their possible roles in the HCV replication by siRNA.The inhibition of G3BP1 expression,a 5'(-)NCR binding protein,caused a marked reduction of HCV both in RNA and protein level (about 55%in RNA level and 57%in protein level when compared with the control), indicating its important role in HCV replication.To study the role of G3BP1 in HCV replication,we first confirmed the interaction between G3BP1 and 5'(-)NCR,and mapped the binding side within the 5'(-)NCR,by RNA pull-down assays.Next,G3BP1 C terminal truncated mutants were used to examine their binding abilities with the HCV 5'(-)NCR RNA.The results indicated that the C terminal RNA Recognition Motif(RRM) and RGG (arginine glycine-rich) boxes of G3BP1 were required for its interaction with HCV 5'(-)NCR RNA.To confirm the hypothesis that G3BP1 was a component of HCV replication complexes(HCV RC),and might interact with 5'(-)NCR in vivo,first we performed co-immunoprecipation assays and the results revealed that G3BP1 could bind NS5B only,but not NS3 and NS5A,when expressed individually in Huh7 cells;but could precipitated all these three HCV RC components in replicon cells.Then confocal microscopy analysis found that G3BP1 was co-localized with the HCV RC protein NS5A in replicon cells and their co-localization disappeared when cells were pretreated by mβCD to destroy the RC.Further more we found that G3BP1,like HCV RC protein NS5A,could also resist to the proteinase K digestion in replicon cells.All these results indicated that G3BP1 was a component of HCV replication complexes. To further study the role of G3BP1 in HCV replication,siRNA was used.Both colony formation assay of replicon cells and HCVcc infection assay showed that although si-G3BP1 had no effect on HCV translation,it could greatly impair the HCV replication after the expression of G3BP1 was significantly inhibited,indicating the essential role of G3BP1 in HCV replication.Then several truncated mutants of G3BP1 were used to examine their functions on HCV replication in both replicon and HCVcc systems.The results indicated that the C terminal RNA Recognition Motif(RRM) and RGG(arginine glycine-rich) boxes of G3BP1 were essential for HCV replication.Finally the time course quantitative assays were performed to monitor the accumulation of both positive and negative strands RNA of HCV in the siG3BP1 treated cells after subgemonic replicon RNA transfection or HCVcc infection.The results suggested that G3BP1 might facilitate the synthesis of HCV minus strain RNA.The above results suggest that the HCV 3'NCR RNA binding proteins,especially G3BP1,may be the important components of HCV RC,and necessary for HCV replication.Our findings may facilitate the understanding of HCV replication as well as the development of novel antiviral drugs.